Most eukaryotic pre-mRNAs contain introns that must be efficiently and accurately spliced out to allow production of functional proteins. The splicing of many pre-mRNAs is further complicated in that the cell must distinguish not only between intron and exon sequences, but also between alternatively spliced exons. This proposal seeks to further understand the mechanisms by which alternatively spliced introns are removed using the a- tropomyosin (a-TM) gene as a model system. In particular, the focus will be on the regulated splicing of a-TM exons 2 and 3 where exon 3 is used in all cells except smooth muscle cells which retain exon 2. A repeated RNA sequence element surrounding exon 3 has been mapped that is responsible for negatively regulating the splicing of this exon in smooth muscle cells. This proposal seeks to determine the identity of this protein and determine its role in regulating alternative splicing. While it is possible that the protein will be specific to smooth muscle, it is also possible that previously identified splicing factors might affect the splicing of a-TM exon 3. Thus, four mammalian constitutive splicing factors (PTB, PSF, hnRNP A1, and U2AF) will be tested for their ability to alter the splicing of a-TM in vivo and in vitro. Each of these proteins binds the polypyrimidine tract which has been shown to be the major determinant in the default selection of exon 3. The binding of each of these proteins to the polypyrimidine tract will be determined as well as a functional analysis of polypyrimidine strength. For PSF, further domain analysis will be carried out to map the functional regions of this newly identified essential pre-mRNA splicing factor.